At present we have only limited information available on the extent to which the chain length of folylpolyglutamates serves to direct the flux of one carbon units through competing pathways in mammalian cells. The studies described herein are aimed at providing basic information on the specificities of various folate-dependent enzymes for the number of glutamyl residues in the polyglutamate "tail" of folate cosubstrates and inhibitors. Such studies can be used to predict patterns of metabolic flux for folate derivatives of differing polyglutamate chain length. They are also useful for predicting the effect of polyglutamylation of clinically important antifolate drugs such as methotrexate on the various folate-dependent reactions. In addition, we believe that folylpolyglutamate substrates may prove very useful in mechanistic studies, particularly where they lead to changes in the order of addition of substrates as compared to the order of addition observed with monoglutamate folate substrate. In the present proposal we plan to continue our examination of the interactions of folylpolyglutamates with folate-dependent enzymes using a combination of steady-state kinetic studies and structural studies. We will continue studies on methylenetetrahydrofolate dehydrogenase, thymidylate synthase, serine hydroxymethyl-transferase and methylenetetrahydrofolate reductase, and will initiate studies on AICAR and GAR transformylase and methionine synthase.